Furthermore, absolute and relative tumor weight showed significant decreases in the PTW and gefitinib groups compared to the TB control (p<0.01, Figure 4E and 4F). However, compared to TB control, absolute and relative weight was significantly increased in PTW groups (p<0.05), but not in gefitinib. There were no differences in serum and spleen cytokine levels between TB control and gefitinib groups.
On the other hand, the number of tumor cells was obviously reduced in the PTW and gefitinib groups. Cells immunoreactive for caspase-3 and poly (ADP-ribose) polymerase (PARP) as apoptotic agents were observed more in the PTW and gefitinib groups than in the TB control group. In contrast, immunoreactive cells for Ki-67 as a marker of proliferation were observed less in the PTW and gefitinib groups.
Angiogenesis in the stroma was assessed by immunostaining for cluster of differentiation (CD)31, and immunoreactive cells were significantly reduced in the PTW and gefitinib groups compared to the TB control (p<0.01). Immunoreactive cells were observed clearly more in the PTW groups than in the TB control group. The TB control group showed atrophic changes with a reduced number of lymphoid cells in the splenic white pulp and lymph nodes (Figure 8).
Interestingly, the decreased parameters in the spleen, lymph nodes and adipose tissue were significantly increased in the PTW groups compared to the TB control group, but not for the gefitinib group (p<0.01).
Discussion
The gradient of the inhibition curve appeared to be more pronounced in PTW treatment than gefitinib, suggesting high sensitivity to PTW in EGFR-TKI-resistant NSCLC. Furthermore, oral administration of PTW inhibited progressive tumor growth showing reduction in tumor size and weight in the NSCLC xenograft mice model. Oxidative stress levels increase in advanced stages of lung cancer, through oncogene activation and subsequent cellular metabolism, while levels of antioxidant molecules decrease [54].
The systemic antitumor effects of PTW are more difficult to interpret because the only small amounts of RONS in administered PTW are expected to reach the tumor via the gastrointestinal tract. However, immunostaining data for caspase-3, PARP, Ki-67, and CD31 further supported the antitumor effects of PTW by increasing apoptosis and inhibiting cell proliferation and stromal angiogenesis in the tumor mass. Excessive oxidative stress on antioxidant mechanisms in the tumor itself is symbolized as an alternative and promising therapeutic options for treatment-refractory tumors for NSCLC [55,56].
The current results in the NT and 4HNE immunostains represented a significant increase in oxidative stress in the PTW groups, as well as in the geftinib compared to the TB control. In this text, we used a gefitinib as a reference drug for the oxidative stress-mediated antitumor effects compared to PTW in chemoresistant NSCLC. The histopathological analyzes supported the improvement of cachexia with PTW treatment, demonstrating the reduced atrophic changes in the spleen and lymph nodes and the increased expression of iNOS and TNF-α in the tumor mass.
The improved immune responses in the PTW groups may be linked to the significant increases in the splenic and peritoneal NK cells, which contribute to the antitumor effects and then improve the cachexia. Together with splenic hypertrophy in the above-mentioned toxicological studies, the results suggest that the antitumor effects of PTW may be mediated by the immune stimulation involved in the oxidative stress [42]. However, the cachexia and immune responses were not different in the gefitinib group compared to the TB control, despite significant inhibition of tumor growth.
This suggests the establishment of new therapeutic strategies using oxidative stress-mediated mechanisms for the treatment of the refractory NSCLC. The extracellular RONS produced in PTW undergoes multiple reactions, as listed below [ 77 ], which play an essential role in cancer cell death by oxidative stress. However, more research will be needed to elucidate the exact mechanisms and specify the role of other antioxidant defense mechanisms, which are not yet included in the current hypothesis.
Conclusions
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Arrows indicate lymphoid follicles in the spleen and lymph node, and the dashed line is for a row of the adipose tissue.
